The present invention provides novel methods for gene delivery and expression in areas that are currently inaccessible through the use of conventional direct protein delivery techniques. In particular, the methods and related products provided herein can be used in the treatment of pulmonary disorders and delivery of anti-viral proteins.
The following review of the background of the invention is merely provided to aid in the understanding of the present invention and neither it nor any of the references cited within it are admitted to be prior art to the present invention.
There are presently several approaches being studied for delivering genes to humans. These approaches have either: (i) removed somatic cells, permanently transformed them in vitro using retrovirus vectors, and reinfused the transformed cells; or (ii) used viruses to deliver the gene. Such therapy has been designed for use with patients having inherited deficiency of gene products, such as proteins, due to abnormalities of the gene during development. Such therapies have also been used with disorders such as emphysema, wherein it is thought that the disease process is a result of a relative deficiency of an antiprotease over a long period of time. Further, diseases such as acute lung injury resulting in the adult respiratory distress syndrome (ARDS) are thought to involve a relative deficiency of antiprotease activity. In addition, cystic fibrosis (CF) is the most common lethal genetic disease in Caucasians (Boat, T. F. et al., The Metabolic Basis of Inherited Disease 2649-2680, 1989). Even though CF can affect several organ systems, almost all patients develop chronic obstructive pulmonary disease and chronic pulmonary infections with resultant respiratory failure and early death.
Normally, the lung contains sufficient quantities of serine antiproteases, principally xcex11 antitrypsin (xcex11AT), to combat the effects of toxic substances involved in such diseases, such as neutrophil elastase (NE). However, in CF and other neutrophil-dominated inflammatory lung diseases, the antiprotease defense system fails to prevent proteolytic damage to lung tissue (Boat, T. F. et al., The Metabolic Basis of Inherited Disease 2649-2680, 1989; Richman-Eisenstat, J. B. Y. et al., Am. J. Phys. 264: L413-L418, 1993).
One attempt at solving some of the above problems is described in International Patent Publication WO 92/19730 (hereby incorporated in its entirety, including any drawings) which describes means for the delivery of a gene encoding human xcex11 antitrypsin to the lungs for expression of the human xcex11 antitrypsin capable of alleviating the enzyme deficiency. Further advances regarding cationic liposome mediated antiprotease gene transfer to reduce the infectivity of RSV in cultured cells is reported in M. Persmark et al., J. Investig. Med. 43 S:220, 1995 Other attempts to deliver particular genes to cells of the lung or airway are described in International Patent Applications with publication numbers WO 93/12756 and WO 93/12240, both of which are incorporated herein by reference in their entirety including any drawings.
Despite the progress and success that has been achieved by such attempts, there still remains a need for a general method to provide gene delivery and expression to areas currently inaccessible to direct protein delivery. See R. C. Hubbard, et al. P.N.A.S. 86:680-684, 1989 (describing attempt to deliver protein via an aerosol and stating xe2x80x9cIn the present study, ≈{fraction (1/500)}th of the administered dose was recoverable in lung lymph, a value likely too low to provide adequate protection for xcex11-antitrypsin deficiencyxe2x80x9d). Such obstacles have previously prevented the successful in vivo treatment of alpha antitrypsin (hereinafter xe2x80x9cAATxe2x80x9d) related disorders such as congenital AAT deficiency, as well as other disorders related to other proteins (for example, prostaglandin (hereinafter xe2x80x9cPGxe2x80x9d) synthase see U.S. patent application Ser. No. 08/459,493, filed Jun. 2, 1995 incorporated herein by reference in its entirety including any drawings) that could not previously be treated by direct protein delivery to the desired target area using conventional methods of protein delivery.
The present invention is based in part on the surprising discovery that certain genes (preferred are genes encoding antiproteases such as AAT) can be delivered and expressed in vivo to certain target areas in animals (preferably mammals, more preferably humans) which have previously been inaccessible (i.e., an insufficient amount or inappropriate form of the protein is able to be provided to give a therapeutic response) to direct protein delivery.
A significant and unexpected advantage of the present invention is the ability of the delivered gene to provide a therapeutic response in situations where direct delivery of the protein (even when delivered at several fold higher serum concentrations) does not produce a therapeutic effect. In particular, delivery of the AAT gene has been found to essentially eliminate endotoxin induced increase in pulmonary vascular resistance, even when serum protein levels are approximately 20 fold lower than produced by protein delivery, which, (in contrast) produces no discernable effect on endotoxin response. In addition, delivery of the AAT gene is able to prevent respiratory syncytial virus infection of lung epithelial cells, where direct delivery of the encoded protein does not. Thus, such methods provide a method for successfully treating disorders caused by a deficiency of the product encoded by the gene of interest in the target area in the particular organism suffering from such a disorder.
The xcex11-antitrypsin protein is the major antiprotease in the lungs of humans. It is believed that both acute lung injury associated with inflammation and emphysema are a consequence of protease/antiprotease imbalance and increasing the antiprotease activity in the lungs is one possible approach to prevention and therapy of these conditions. In addition, there is a genetic form of xcex11-antitrypsin deficiency where patients develop emphysema at an early age.
Patients with xcex11-antitrypsin deficiency are now being treated with intravenous administration of the xcex1-antitrypsin protein. This intervention is expensive, requires relatively frequent intravenous infusions, can cause reactions in the patient and is of unproven efficacy. In addition, since the protein is derived from human blood products, a risk of infection by contaminating viruses, such as HIV is present. Gene therapy by delivery of the DNA encoding xcex11-antitrypsin to the airway cells could provide a less invasive, safer, cheaper and more effective therapy.
Thus, in one aspect the invention provides a method for delivering a nucleic acid molecule to a location in an animal that is inaccessible to direct protein delivery. Those skilled in the art will understand that several conventional methods for directly delivering proteins exist and are commonly used in the art. The method involves the step of administering a positively charged liposome to the animal. The positively charged liposome is associated with the nucleic acid molecule, wherein said nucleic acid molecule is in operable association with a promoter. Those skilled in the art will recognize that a wide variety of promoters may be used to assist in targeting the desired location.
In preferred embodiments, the nucleic acid molecule encodes human xcex11 antitrypsin, the location is selected from the group consisting of an endothelial lung cell, a smooth muscle cells adjacent to the endothelial lung cell, and lung parenchyma, or the location is selected from the group consisting of a liver cell, a muscle cell, an osteogenic cell, synoviocyte, and a lung cell. Other preferred locations and genes are shown in Table I below.
In other preferred embodiments the animal is a mammal, preferably a human, and the positively charged liposome is Lipofectin(trademark). Other suitable liposomes are described in International Patent Application with publication numbers WO 93/12756 and WO 93/12240, both of which are incorporated by reference in their entirety, including any drawings. Preferably the nucleic acid sequence encodes a therapeutically effective protein (e.g., an antiprotease) and the method further involves expressing the nucleic acid sequence to provide the protein to the location.
In another aspect, the invention provides a method for preventing or treating an animal having a disorder. At least one symptom associated with the disorder is caused at least in part by an insufficient amount or form of protein in a particular location of the animal. The method involves the step of delivering a gene encoding the protein to the location and expressing the gene. Preferably at least 10%, more preferably at least 50%, most preferably at least 90% of the nucleic acid that is administered is delivered to said location.
In another aspect the invention provides a method for delivering a nucleic acid molecule to a location in an animal. The method involves the step of administering a positively charged liposome to the animal. The positively charged liposome is associated with the nucleic acid molecule, and the nucleic acid molecule is in operable association with a promoter. Delivery of the gene is capable of producing a therapeutic response, but direct delivery of the protein encoded by the gene does not produce a therapeutic response.
In preferred embodiments, delivery of the gene produces a therapeutic response even when delivered at a 10-fold lower serum concentration than the protein (which does not produce a therapeutic response when delivered directly as a protein). Preferably, the therapeutic response is elimination of an endotoxin induced increase in pulmonary vascular resistance.
In another aspect, a method of producing an elevated therapeutic response relative to the therapeutic response created by direct delivery of a protein. The method involves the step of delivering a nucleic acid molecule encoding the protein.
In preferred embodiments, the therapeutic response created by direct delivery of the protein is non-existent or immeasurable, and the protein is an antiprotease. Preferably, the enhanced therapeutic response is created in a patient suffering from a disorder selected from the group consisting of adult respiratory distress syndrome, cystic fibrosis, respiratory syncytial virus infection, and chronic obstructive pulmonary disease.
The composition is preferably capable of delivering the nucleic acid or oligonucleotide into a cell. By xe2x80x9cdelivering the nucleic acid or oligonucleotide into a cellxe2x80x9d is meant transporting a complexed and condensed nucleic acid or a complexed oligonucleotide in a stable and condensed state through the membrane of a cell (in vitro or in vivo), thereby transferring the nucleic acid or oligonucleotide from the exterior side of the cell membrane, passing through the lipid bilayer of the cell membrane and subsequently into the interior of the cell on the inner side (i.e., cytosol side) of the cell membrane and releasing the nucleic acid or oligonucleotide once within the cellular interior.
In a preferred embodiment at least 1% of the nucleic acid or oligonucleotide in the composition is delivered into the cell or cells of the desired target location. In a more preferred embodiment, at least 10% of the nucleic acid or oligonucleotide is so delivered. In an even more preferred embodiment, at least 50% of the nucleic acid or oligonucleotide is so delivered. In a most preferred embodiment, at least 90% of the nucleic acid or oligonucleotide is so delivered.
Furthermore, the composition may prevent lysosomal degradation of the nucleic acid by endosomal lysis. In addition, although not necessary, the composition may also efficiently transport the nucleic acid through the nuclear membrane into the nucleus of a cell.
By xe2x80x9cnucleic acidxe2x80x9d is meant both RNA and DNA including: cDNA, genomic DNA, plasmid DNA, antisense molecule, polynucleotides or olignucleotides, RNA or mRNA. In a preferred embodiment, the nucleic acid administered is plasmid DNA which comprises a xe2x80x9cvectorxe2x80x9d.
By xe2x80x9cvectorxe2x80x9d is meant a nucleic acid molecule incorporating sequences encoding polypeptide product(s) as well as, various regulatory elements for transcription, translation, transcript stability, replication, and other functions as are known in the art and as described herein. Vector can include expression vector.
An xe2x80x9cexpression vectorxe2x80x9d is a vector which allows for production or expressing a product encoded for by a nucleic acid sequence contained in the vector. The product may be a protein or a nucleic acid such as an mRNA which can function as an antisense molecule.
A xe2x80x9ctranscript stabilizerxe2x80x9d is a sequence within the vector which contributes to prolonging the half life (slowing the elimination) of a transcript.
A xe2x80x9cDNA vectorxe2x80x9d is a vector whose native form is a DNA molecule. By xe2x80x9cnon-viralxe2x80x9d is meant any vector or composition which does not contain genomic material of a viral particle.
An xe2x80x9cantisense moleculexe2x80x9d can be a mRNA or an oligonucleotide which forms a duplex with a complementary nucleic acid strand and can prevent the complementary strand from participating in its normal function within a cell. For example, expression of a particular growth factor protein encoded by a particular gene.
A xe2x80x9cgene productxe2x80x9d means products encoded by the vector. Examples of gene products include mRNA templates for translation, ribozymes, antisense RNA, proteins, glycoproteins, lipoproteins and phosphoproteins.
xe2x80x9cPost-translational processingxe2x80x9d means modifications made to the expressed gene product. These may include addition of side chains such as carbohydrates, lipids, inorganic or organic compounds, the cleavage of targeting signals or propeptide elements, as well as the positioning of the gene product in a particular compartment of the cell such as the mitochondria, nucleus, or membranes. The vector may comprise one or more genes in a linear or circularized configuration. The vector may also comprise a plasmid backbone or other elements involved in the production, manufacture, or analysis of a gene product. The nucleic acid may be associated with a targeting ligand to effect targeted delivery.
A xe2x80x9ctargeting ligandxe2x80x9d is a component of the delivery system or vehicle which binds to receptors, with an affinity for the ligand, on the surface or within compartments of a cell for the purpose of enhancing uptake or intracellular trafficking of the vector. Glucans such as Tris-galactosyl residues, carnitine derivatives, mannose-6-phosphate, monoclonal antibodies, peptide ligands, and DNA-binding proteins represent examples of targeting ligands which can be used to enhance uptake.
xe2x80x9cIntracellular traffickingxe2x80x9d is the translocation of the vector within the cell from the point of uptake to the nucleus where expression of a gene product takes place. Alternatively, cytoplasmic expression of a nucleic acid construct utilizing, for example, a T7 polymerase system may be accomplished. Various steps in intracellular trafficking include endosomal release and compartmentalization of the vector within various extranuclear compartments, and nuclear entry.
xe2x80x9cEndosomal releasexe2x80x9d is the egress of the vector from the endosome after endocytosis. This is an essential and potentially rate limiting step in the trafficking of vectors to the nucleus. A lytic peptide may be used to assist in this process.
A xe2x80x9clytic peptidexe2x80x9d is a peptide which functions alone or in conjunction with another compound to penetrate the membrane of a cellular compartment, particularly a lysosomal or endosomal compartment, to allow the escape of the contents of that compartment to another cellular compartment such as the cytosolic and/or nuclear compartment.
xe2x80x9cCompartmentalizationxe2x80x9d is the partitioning of vectors in different compartments within a defined extracellular or intracellular space. Significant extracellular compartments may include, for example, the vascular space, hair follicles, interstitial fluid, synovial fluid, cerebral spinal fluid, thyroid follicular fluid. Significant intracellular compartments may include endosome, potosome, lysosome, secondary lysosome, cytoplasmic granule, mitochondria, and the nucleus.
xe2x80x9cNuclear entryxe2x80x9d is the translocation of the vector across the nuclear membrane into the nucleus where the gene may be transcribed.
xe2x80x9cEliminationxe2x80x9d is the removal or clearance of materials (vectors, transcripts, gene products) from a specific compartment over time. This term may be used to reflect elimination from the body, the vascular compartment, extracellular compartments, or intracellular compartments. Elimination includes translocation (excretion) from a particular compartment or biotransformation (degradation).
The compounds which increase the efficacy of transfection of a nucleic acid are suitable for internal administration. By xe2x80x9csuitable for internal administrationxe2x80x9d is meant that the compounds are suitable to be administered within the tissue of an organism, for example within a muscle or within a joint space, intradermally or subcutaneously. Other forms of administration which may be utilized are topical, oral, pulmonary, nasal and mucosal; for example, buccal, vaginal or rectal. These substances may be prepared as solutions, suspensions, gels, emulsions or microemulsions. Oil suspensions of lyophilized nucleic acid, such as plasmid DNA may be utilized. Delivery systems for these oil suspensions include, but are not limited to, sesame oil, cottonseed oil, soybean oil, lecithins, Tweens, Spans and Miglyols.
By xe2x80x9csolutionsxe2x80x9d is meant water soluble substances and/or surfactants in solution with nucleic acids. By xe2x80x9csuspensionsxe2x80x9d is meant water insoluble oils containing suspended nucleic acids. By xe2x80x9cgelsxe2x80x9d is meant high viscosity substances containing nucleic acids. By xe2x80x9cemulsionxe2x80x9d is meant a dispersed system containing at least two immiscible liquid phases. Emulsions usually have dispersed particles in the 0.1 to 100 micron range. They are typically opaque and thermodynamically unstable. Nucleic acids in the water phase can be dispersed in oil to make a w/o emulsion. This w/o emulsion can be dispersed in a separate aqueous phase to yield a w/o/w emulsion. Alternatively, a suitable oil could be dispersed in an aqueous phase to form an o/w emulsion.
A xe2x80x9cmicroemulsionxe2x80x9d has properties intermediate to micelles and emulsions and is characterized in that they are homogenous, transparent and thermodynamically stable. They form spontaneously when oil, water, surfactant and co-surfactant are mixed together. Typically, the diameter of the dispersed phase is 0.01 to 0.1 microns, usually of the w/o and o/w type. The sustained-release compound containing a nucleic acid is administered to the tissue of an organism, for example, by injection. In one embodiment the tissue is preferably muscle tissue. In another embodiment the tissue is preferably a joint space.
By xe2x80x9csustained-release compoundxe2x80x9d is meant a substance with a viscosity above that of an isotonic saline solution (150 mM NaCl) containing a nucleic acid; for example, DNA in saline at 1 mg/ml has a viscosity of 3.01 mPaxc2x7sec, DNA in saline at 2 mg/ml has a viscosity of 3.26 mPaxc2x7sec, DNA in saline at 3 mg/ml has a viscosity of 5.85 mPaxc2x7sec (Viscosity measurements were performed at 25xc2x0 C. in a Brookfield DV-III Rheometer with a No. 40 Spindle at 75 rpm for 30 minutes). Preferably the sustained-release compound has a viscosity in the range of about 0.1-20,000 mPaxc2x7sec above that of a complexation in which isotonic saline is the delivery system for a nucleic acid. More preferably the range is about 0.1-5000 mPaxc2x7sec above that of a complexation in which isotonic saline is the carrier for a nucleic acid. Even more preferably the range is about 0.1-1000 mpaxc2x7sec above that of a complexation in which isotonic saline is the carrier for a nucleic acid.
xe2x80x9cTargeted deliveryxe2x80x9d involves the use of targeting ligands which specifically enhance translocation of a nucleic acid to specific tissues or cells. A xe2x80x9ctargetxe2x80x9d is a specific organ, tissue, or cell for which uptake of a vector and expression of a gene product is intended. xe2x80x9cUptakexe2x80x9d means the translocation of the vector from the extracellular to intracellular compartments. This can involve receptor mediated processes, fusion with cell membranes, endocytosis, potocytosis, pinocytosis or other translocation mechanisms. The vector may be taken up by itself or as part of a complex. xe2x80x9cBindingxe2x80x9d is an intermediate step in uptake of some compositions involving a high-affinity interaction between a targeting ligand and a surface receptor on a target cell.
By xe2x80x9coligonucleotidexe2x80x9d is meant a single-stranded polynucleotide chain. In a preferred embodiment, the oligonucleotide is less than 100 residues in length. In a more preferred embodiment, the oligonucleotide is less than 50 residues in length. In a most preferred embodiment, the oligonucleotide is less than 30 residues in length.
In a preferred embodiment, the invention features a composition capable of complexing and condensing the nucleic acid or oligonucleotide. These compositions provide smaller, or condensed, and more stable nucleic acid particles for delivery, thereby enhancing the transfection rate of nucleic acid into the cell and the subsequent expression therein.
By xe2x80x9ccomplexingxe2x80x9d is meant a high affinity interaction, based upon non-covalent binding, between the chitosan-based substance and the nucleic acid or oligonucleotide. By xe2x80x9caffinityxe2x80x9d is meant the selective tendency of elements to combine with one, rather than another element, when the physicochemical conditions are appropriate. This interaction is most preferably an ionic interaction but may be brought about wholly or in part by hydrogen bonding, Van der Walls interactions or other chemical attractions commonly recognized by those in the art. The compounds which complex and condense a nucleic acid may also interact or associate with the nucleic acid by intermolecular forces and/or valence bonds such as: Van der Waals forces, ion-dipole interactions, ion-induced dipole interactions, hydrogen bonds, or ionic bonds.
These interactions may serve the following functions: (1) Stereo selectively protect nucleic acids from nucleases by shielding; (2) facilitate the cellular uptake of nucleic acid by xe2x80x9cpiggyback endocytosisxe2x80x9d. By xe2x80x9cpiggyback endocytosisxe2x80x9d is meant the cellular uptake of a drug or other molecule complexed to a delivery system that may be taken up by endocytosis (C. V. Uglea and C. Dumitriu-Medvichi, Medical Applications of Synthetic Oligomers. In: xe2x80x9cPolymeric Biomaterials.xe2x80x9d Edited by Severian Dumitriu. Marcel Dekker, Inc. 1993) and incorporated herein by reference including all drawings and figures. To achieve the desired effects set forth, it is desirable, but not necessary, that the substances which condense and complex nucleic acid have amphipathic properties; that is, the substance has both hydrophilic and hydrophobic regions. The hydrophilic region of the substance may associate with the largely ionic and hydrophilic regions of the nucleic acid, while the hydrophobic region of the substance may act to retard diffusion of nucleic acid and to protect nucleic acid from nucleases. Additionally, the hydrophobic region may specifically interact with cell membranes, possibly facilitating endocytosis of the composition and thereby nucleic acid associated with the compound. This chitosan-based composition may increase the pericellular concentration of nucleic acid.
By xe2x80x9ccondensingxe2x80x9d is meant charge neutralization, exclusion of water and compacting into colloidal particles. The composition which condense and complex nucleic acid may also achieve one or more of the following effects, due to their physical, chemical or rheological properties: (1) Protect nucleic acid, for example plasmid DNA, from nucleases; (2) increase the area of contact between nucleic acid, such as plasmid DNA, through extracellular matrices and over cellular membranes, into which the nucleic acid is to be taken up; (3) concentrate nucleic acid, such as plasmid DNA, at cell surfaces due to water exclusion; (4) indirectly facilitate uptake of nucleic acid, such as plasmid DNA, either increasing interaction with cellular membranes and/or by perturbing cellular membranes due to osmotic, hydrophobic or lytic effects.
By xe2x80x9cincrease the efficacy of transfectionxe2x80x9d is meant that a nucleic acid or oligonucleotide when administered to an organism in a composition comprising such a substance will be more readily taken up into the interior of a cell by translocating across the cellular membrane than if administered in a composition without such a substance, for example when administered in a formulation such as a saline solution. The increased efficiency of uptake of nucleic acid, or oligonucleotide into cells could occur, for example, due to a better steric fit between the composition containing the nucleic acid and a pit on the surface of the cellular membrane or due to protection of the nucleic acid from attack by nucleases.
In another preferred embodiment, the composition has a net positive charge ratio. By xe2x80x9cnet chargexe2x80x9d is meant the resulting positive, negative or neutral character of a compound which is determined after balancing the total number of positive and negative charges possessed by a molecule or compound. For example, the DNA molecule, has a net negative charge due to the presence of two anionic phosphate moieties on each base pair of the molecule. The number of negatively charged phosphates exceed in number the total number of positive charges on the DNA molecule. Thus the surfeit of negative charges imparts a net negative character or charge to DNA. The number of negative charges to positive charges on compositions determines the net charge ratio. The net charge ratio is symbolized by (xe2x88x92/+) where a dash, xe2x80x9cxe2x88x92xe2x80x9d, stands for a negative charge and a plus sign, xe2x80x9c+xe2x80x9d, stands for a positive charge. A net charge ratio of 1:1(xe2x88x92/+) is neutral; of 2:1(xe2x88x92/+) is negative and of 1:2(xe2x88x92/+) is positive.
Another embodiment features the composition additionally mixed with a cryoprotectant. By xe2x80x9ccryoprotectantxe2x80x9d is meant any chemical or compound which will serve to protect nucleic acid and oligonucleotides and the complexed particles during lyophilization, storage, and subsequent rehydration. Examples of xe2x80x9ccryoprotectantsxe2x80x9d include, but are not limited to, such compounds as lactose, sucrose, mannitol, and trehalose.
In another aspect, the nucleic acid or oligonucleotide is delivered to a cell by the step of exposing the composition to the cell. The method may be performed in vitro, in vivo, or on a cell that has been removed from a living organism. If the method is performed in vivo, then the exposing step may be performed by administering the composition to an organism.
By xe2x80x9cadministeringxe2x80x9d is meant the route of introduction of the composition into a body. Administration can be directly to a target tissue or through systemic delivery. In particular, administration may be by direct injection to the cells. Routes of administration include, but are not limited to, intramuscular, aerosol, oral, topical, systemic, nasal, ocular, intraperitoneal and/or intratracheal, buccal, sublingual, oral, intradermal, subcutaneous, pulmonary, intra-artricular, and intra-arterial. In a preferred embodiment administration is by intravenous administration.
By xe2x80x9corganismxe2x80x9d is meant a living entity capable of replication. In a preferred embodiment the organism is an animal, in a more preferred embodiment a mammal, and in a most preferred embodiment a human.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims.